The objectives of the present study were (1) to evaluate the effect of prepartum cholecalciferol treatment on serum Ca concentration during the first 10 d after calving and (2) to evaluate the effect of treatment on subsequent health and performance. Multiparous Holstein cows (n = 377) from one dairy farm were fed a negative dietary cation-anion difference diet (−31 mEq/kg of DM) for the last 21 d of gestation. On d 275, the animals were randomly assigned to a control or a treatment group. Cows in the control group were left untreated, and cows in the treatment group received an injection of 12 × 106 IU of cholecalciferol intramuscularly on the day of enrollment. If treated cows did not deliver the calf within 6 d, they were reinjected with 10 × 106 IU of cholecalciferol. Blood samples were drawn on 1, 2, 3, 5, 7, and 10 days in milk (DIM) and analyzed for serum Ca, P, and Mg concentrations. In a subsample of cows (50 control cows, 35 cows treated once with cholecalciferol, and 15 cows treated twice) serum haptoglobin, nonesterified fatty acids, β-hydroxybutyrate, and 25-hydroxycholecalciferol concentrations were analyzed on 1, 5, and 10 DIM. Binary data [retained placenta (RP), metritis] were analyzed using logistic regression models. Repeated measures ANOVA with first-order autoregressive covariance was performed to evaluate the treatment effect on milk yield over the first 10 test days after parturition, 25-hydroxycholecalciferol, serum Ca, P, Mg, β-hydroxybutyrate, nonesterified fatty acids, and haptoglobin concentrations. Cox proportional hazards were used to model the time to event outcomes (time to pregnancy within 200 d, culling until 300 DIM). After enrollment of 31.4% of cows and a preliminary analysis, adverse reactions became apparent, and the study was stopped. Cows treated with cholecalciferol had a greater risk of incurring RP and metritis. The adjusted mean incidences were 2.0%, 7.7%, and 4.0% for RP, and 21.6%, 39.3%, and 33.3% for metritis for control cows, cows treated once, and cows treated twice with cholecalciferol, respectively. Compared with control cows, cows injected once with 12 × 106 IU of cholecalciferol produced less energy-corrected milk on the first (−3.76 kg) and second (−2.75 kg) test days, respectively. Cows injected twice with cholecalciferol (12 × 106 IU of cholecalciferol and 10 × 106 IU 1 wk later) had a reduced milk yield only at first test day (−3.80 kg). Treatment with cholecalciferol led to a significant increase in 25-hydroxycholecalciferol on d 1, 5, and 10 after calving. Serum Ca and P concentrations were significantly increased in cows treated with cholecalciferol, but serum Mg concentrations were significantly reduced. Haptoglobin concentrations were significantly increased on 5 DIM in cows injected once with 12 × 106 IU of cholecalciferol. Although we observed no effect of treatment on culling until 300 DIM, time to pregnancy was delayed by 34 d in cows injected once with 12 × 106 IU of cholecalciferol. In the present study, injection with 12 × 106 IU of cholecalciferol had detrimental effects on health and milk production despite the beneficial effects on Ca homeostasis.